The invention relates to phase supports for the partition chromatography of macromolecules, in particular in an aqueous polyethylene glycol/dextran two-phase system, to a process for their preparation and to their use.
It is known that biological macromolecules, subcellular units, bacteria and eucaryotic cells can be separated by counter-current partition in an aqueous polyethylene glycol/dextran system (P. .ANG.. Alberston "Partition of Cell Particles and Macromolecules" (1971), 2nd edition, Almquist and Wiksell, Stockholm). These counter-current partition processes are, however, extremely involved in terms of apparatus and are time-consuming, especially if, in the case of small partition coefficients, a large number of counter-current partition stages is necessary in order to achieve the desired separation. Attempts have therefore already been made to replace this counter-current partition process by a partition chromatography process, since it is possible to achieve a large number of separation stages considerably more easily in this manner. However, these attempts have hitherto failed owing to a lack of suitable supports for the stationary phase.
Hitherto it has only been possible to separate by chromatography double-stranded nucleic acids in an aqueous polyethylene glycol/dextran system using cellulose as the support for the dextran-rich stationary phase (W. Muller, H. J. Schuetz, C. Guerrier-Takada, P. E. Cole and R. Potts, Nucleic Acids Research, Volume 7, No. 8 (1979), 2483 to 2499, and W. Muller and G. Kutemeier, Eur. J. Biochem. 128 (1982), 231 to 238). In these investigations of the liquid/iquid chromatography of DNA fragments, the support materials employed for the dextran-rich phase of the aqueous polyethylene glycol/dextran system were a number of materials of which cellulose, in particular, has proved suitable, since it exhibits an adequate affinity for the dextran-rich phase. However, these phase supports cannot be used for proteins and protein-containing cell constituents because of their pronounced adsorption properties; troublesome effects caused by adsorption also manifest themselves in the case of ribonucleic acids. Although cationic or anionic gels based on polysaccharides bind the dextran-rich phase as well as cellulose does, they can only be used for isocratic separation processes, since the phase is repelled as soon as the electric phase potential changes in the course of the gradient elution with various salts in the mobile, polyethylene-rich phase.
Amongst the neutral gels which would be suitable for use as potential phase supports for partition chromatography of this type are porous copolymers based on vinyl compounds (Fractogels made by Merck, Darmstadt) and the polyacrylamide gels (Biogels made by Biorad). The former bind too small an amount of the dextran phases for general use, whereas in the latter case the bound phase is hardly accessible to macromolecules. This state of affairs is also clearly emphasised in the literature reference quoted above, Eur. J. Biochem. 128 (1982), page 233. This inaccessibility of the bound phase also applies to polyacrylamide/agarose combination gels (for example the AcA-Ultrogels made by IDF).